The objective of the proposed research is to characterize the RNA splicing reaction by which intervening sequences are removed from RNA molecules in eukaryotic organisms. In the amplified ribosomal RNA genes of the ciliated protozoan Tetrahymena thermophila, the 26S rRNA coding region is interrupted by a 400 base pair intervening sequence. This sequence is contained in the primary transcript but is subsequently deleted from the RNA by splicing. We have recently developed an in vitro splicing system that permits the isolation of the excised 400 nucleotide RNA fragment ("IVS RNA"). The splicing reaction will be characterized in vitro in isolated nuclei as well as in vivo to determine the nature of the reaction products and the kinetics of their synthesis and degradation. Hybridization of nuclear RNA with recombinant DNA containing the intervening sequence will be used to isolate the unspliced pre-rRNA. This material will provide the basis for an assay to detect splicing activity, which should allow purification of the enzyme(s) involved. The enzyme(s) may require an rRNA-protein complex as a substrate, in which case nascent ribosomal particles will be isolated. The number of different enzymes involved in the pre-rRNA splicing reaction will be determined. The subunit structure, cofactor requirements, and specificity of each enzyme will be investigated. Temperature-sensitive mutants of Tetrahymena that are known to accumulate pre-rRNA at the nonpermissive temperature will be screened for splicing activity. Splicing mutants would be a valuable source of the unspliced pre-rRNA substrate, and might also help characterize the number of enzymatic steps required for splicing. The Tetrahymena pre-rRNA splicing reaction shares some key features with mammalian mRNA splicing, such as the large size of the intervening sequence and the lack of apparent base-pairing potential near the splice junctions. The Tetrahymena system, however, is much more amenable for detailed study.